FIG 5.

Biotinylated proteins in virus particles. ΔMA-BirA* (lanes A to I) or MA-BirA* (lanes J to R) proviral constructs were transfected into 293T cells alone or along with WT (+WT) or PR⁻ (+PR−) HIV-1 proviral constructs. At 3 days posttransfection, proteins from collected virus particles were electrophoretically separated and subjected to detection of biotinylated proteins with streptavidin (lanes A to C and J to L), viral proteins with an anti-CA antibody (lanes D to F and M to O), or viral proteins with an anti-MA antibody (lanes G to I and P to R). Molecular size markers, as indicated, were run in parallel lanes. PrΔMABirA*, PrMABirA*, PrGag, CA, and MA bands are as indicated. To match figure sizes of streptavidin and CA blots, portions of MA blots that showed no immunoreactive bands between CA and MA were truncated. As shown, MA-BirA* proteins in viruses were readily detectable, while ΔMA-BirA* virus particle release was inefficient (lanes A and D) in the absence of cotransfected HIV-1 WT or PR⁻ constructs. Relative to WT HIV-1, virus particle release levels for MA-BirA* and ΔMA-BirA* were 70% ± 19% and 15% ± 11%, respectively, while cotransfection with WT HIV-1 increased MA-BirA* normalized release levels to 100% ± 11% and ΔMA-BirA* levels to 92% ± 20%. Note that the PrGag-sized band (band 1) in lane A may correspond to a processed BirA*-CA protein, although it stained marginally with anti-CA, while band 2 (lane A) migrated at the size of the BirA* protein (38 kDa). In the ΔMA-BirA* anti-MA blot, band 3 appears to be an anomaly, because PrΔMABirA* bands were not detected in lanes H and I. For the MA-BirA* virus, bands 4 and 5 had mobilities of 50 kDa and 46 kDa, strongly reacted with the anti-MA antibody (lane P), and appear to correspond to MA-BirA* (band 4) and a processed variant of MA-BirA* (band 5). Note also that band 6 comigrated with CA (lane M) and an anti-MA-reactive band (band 7). Data are representative of the results of nine separate experiments.